BACKGROUND: Biomass prehydrolysates from dilute acid pretreatment contain a considerable amount of fermentable sugars for biofuels production. However, carbonyl degradation compounds present severe toxicity to fermentation microbes. Furans (such as furfural and hydroxymethylfurfural), aliphatic acids (such as acetic acid, formic acid and levulinic acid) and phenolic compounds (such as vanillin and syringaldehyde) have been suggested to be the main inhibitors in biomass prehydrolysates. However, no single compound has been determined as the dominant toxic inhibitor. The effects of various detoxification methods on inhibitors removal have not been fully understood. RESULTS: The effects of overliming and activated carbon (AC) detoxification on the removal of inhibitors and butanol fermentation of the poplar prehydrolysates were investigated. Gas chromatography–mass spectrometry (GC/MS) was used to identify and quantify 46 carbonyl compounds as potential inhibitors. It was observed that overliming and AC treatment alone did not make the prehydrolysates fermentable with Clostridium saccharobutylicum. The sequential overliming and AC resulted in a remarkable fermentability and a high butanol yield at 0.22 g g⁻¹ sugar. The inhibitor removal in the prehydrolysates treated by overliming and AC was also examined by GC/MS. Overliming removed 75.6% of furan derivatives and 68.1% of aromatic monomers. In comparison, AC (5.0% w/v) removed 77.9% of furan derivatives and 98.6% of aromatic monomers. In addition, overliming removed much more 2,5-furandicarboxyaldehyde, 5-ethylfuran-2-carbaldehyde and 2,5-hexanedione than AC did. On the contrary, AC could remove considerably more phenolic acids than overliming. In the sequential detoxification, both dialdehydes/diketones and phenolic acids were extensively removed. This could be the main reason why the sequential detoxification enabled a remarkable ABE fermentation for the prehydrolysates. CONCLUSIONS: This study indicated that the effect of overliming and AC treatment on inhibitors removal was related to their chemical structures. Overliming removed more dialdehydes and diketones than AC treatment, while AC removed more phenolic acids than overliming. Sequential overliming and AC treatment were required to make the prehydrolysates fermentable with C. saccharobutylicum. The study also suggested different detoxification method was needed for ABE fermentation of the prehydrolysate as compared to ethanol fermentation.